Cyclopropyl unsaturated quinoline compound used as leukotriene receptor antagonist and applications thereof

ABSTRACT

A cyclopropyl unsaturated quinoline compound used as leukotriene receptor antagonist and applications thereof, wherein the structural formula of the compound is as follows: 
     
       
         
         
             
             
         
       
     
     An application of the compound or its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs in preparing a drug for treating and/or preventing and/or delaying and/or providing adjuvant therapy for asthma and/or allergic rhinitis and asthma syndromes is provided. A drug composition includes the cyclopropyl unsaturated quinoline compound or the pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. The compound series has favorable foreground in preparing the drugs for treating and/or preventing and/or delaying and/or providing adjuvant therapy for asthma and/or allergic rhinitis and asthma syndromes.

TECHNICAL FIELD

The present invention relates to a cyclopropyl unsaturated quinolinecompound used as leukotriene receptor antagonist and applicationsthereof.

BACKGROUND OF THE INVENTION

Asthma is a chronic inflammation disease characterized by reversibleairway obstruction and non-specific bronchial hyperresponsiveness, andinvolves a complicated process of a series of inflammatory cells andinflammatory mediators. Common symptoms of an asthma patient areparoxysmal asthma, dyspnea, chest distress, or begma, and a few patientsmay have thoracodynia. At present, the asthma is a common disease and afrequently-occurring disease, which severely affects people's physicaland psychological health.

The mechanism of airway obstruction in bronchial asthma (asthma) isrelated to bronchospasm caused by contraction of airway smooth muscle,mucous edema caused by vascular leakage, increased mucus secretion, andinfiltration of inflammation cells induced by eosinophils, and a varietyof inflammatory mediators, such as histamine, leukotrienes (LTs),thromboxane, prostaglandin, or the like, participate in an inflammationreaction of upper and lower airways. The asthma relates to a complicatedprocess of a series of inflammatory cells and inflammatory meditors[British Medical Journal 1998, 316(15):1257-1258]. Researches show thatleukotrienes plays an important role In the pathogenesisof asthma[Sampson A, Holgate S. Leukotriene modifiers in the treatment ofasthma[J]. BMJ, 1998, 316(5): 1257-1258.], and plays an key role on theoccurrence and development of the asthma. Therefore, the leukotrienereceptor antagonist becomes the most effective mediator antagonist inclinic treatment of asthma, and uses of these drugs may accomplish animportant breakout in asthma treatment.

In the later period of 1970s, cysteinyl leukotrienes (CysLT) is found tobe an important mediator of asthma. The leukotrienes is generated byinflammatory cells such as mastocyte, oxyphil cell, or the like, and hasmultiple biological effects in the pathogenes is of asthma:increasingformation of vascular permeability and edema; increasing mucusproduction and reducing mucociliary transportation; attracting theinflammatory cells (such as eosinophils) to migrate from blood to theairway and release the inflammatory mediators, which may damage airwayepithelium; and directly causing bronchospasm and stimulating the smoothmuscle cell proliferation. The leukotrienes acts through the receptorthereof. The leukotriene receptors are distributed in all smooth musclecells, dendritic cells, eosinophils, monocytes, macrophages, and Blymphocytes. An immunofluorescence detection shows that the CysLT1receptors are distributed in both the smooth muscle of a central airwayand a peripheral small airway in a lung tissue. The leukotriene receptorantagonist (LTRA) may improve aeration and relieve symptoms of asthmapatient, which further proves the effect of the leukotrienes for asthma.

As gradually known effects of LTs in the bronchial hyperresponsivenessof asthma and confirmation of the LTs receptor sites, concerns have beenraised about the situation the inhibitor sythesized by leukotrienereceptor antagonist (LTRA) and LTs in prevention and treatment ofasthma. The first leukotriene D4 (LTD4) receptor antagonist ibudilast(ibudilast, ketas) was applied in clinic in 1989. A new-generation LTRAzafirlukast (commercially named as encoreter) was marketed abroad asanti-asthmatic, anti-inflammatory and antiallergic drug in 1996, and hadexperienced certain clinic applications. Drugs with these LTs antagonismeffects are a new trend to treat asthma at currently.

Mechanism of the leukotriene receptor antagonist (LTRAs) mainly lies inthe following aspects: 1. Anti-inflammatory effect, preventing andrelieving infiltration of inflammation cells. After taking zafirlukastor montelukast, as the improvement of lung function, lymphocyte counts,basophilic granulocyte counts, eosinophilic granulocyte counts, andscavenger cell counts in sputum, peripheral blood, and bronchoalveolarlavage fluid (BALF) of the patient are remarkablely reduced, and thereduction degree is related to the improvement degree of vital capacityand daytime symptoms; 2. Relaxing the bronchial smooth muscle; 3.Suppressing bronchus contraction caused by sports. In asthma patients,the bronchus contraction caused by sports is about 70% to 80%, and LTRAscan significantly suppress the bronchus contraction caused by sports;and 4. Effects on aspirin intolerance asthma (AIA), wherein the patientsuffering from aspirin intolerance asthma present characteristics ofchronic rhinitis, repeatedly occurrence of nasal polyp, asthma, andintolerance of the drugs like aspirin, and this asthma is difficult tocontrol by regular treatments, and the patients of this type are oftenaccompanied with over-producing cysteinyl leukotrienes and up regulationof LTC 4 synzyme. Many researches verify that the LTC4 synzyme ofbronchus biopsy tissue of AIA is over expressed accompanying withincreasing cysteinyl leukotriene level in the bronchoalveolar lavagefluid when comparing the patient suffering the aspirin intoleranceasthma with healthy persons.

LTRAs has good tolerance, and large-scale clinical experiments in recentyears verify that compared with a placebo, the LTRAs has significantclinical therapeutic effects, and no apparent side effect have beenfound in long term usage and has good tolerance, which is also veryeffective and safe for children, and no occurrence of death and severenegative reactions have been reported.

The LTRAs available on the market currently and applied in clinic mainlyincludes Zafirlukast (Zafirlukast,

, Acolate), Pranukast (Pranukast), and Montelukast (Montelukast,

, Singulair). Wherein, Montelukast is a novel and powerful blocker drugof cysteinyl leukotriene receptor 1 (CysLTR.) with high selectivity andgood tolerance developed in recent years, which can competitivelyantagonize the combination of leukotrienes D4 (LTD4) and Cys-LT1receptor, thereby to suppress the activity of the leukotrienes, andreduce the expression of endothelium growth factor of blood vessel toadjust the permeability of the blood vessel, and improve the airwayedema. The drug can effectively prevent and suppress the increase ofvascular permeability, bronchospasm, and airway mucus hypersecretioncaused by leukotrienes, and reduce the airway hyperresponsiveness, whichhas no apparent negative effects to important organs or systems, and hasgood compliance. As the inflammation mediator antagonist, itsanti-asthmatic effect has been proved [Markham A, Faulds D. Montelukast[J]. Drugs, 1998, 56(2):251-7.]. The Montelukast is mainly used forprevention and long-term treatment of asthma of adult and child inclinic [LEE K S, KIM S R, PARK H S, et al. Cysteinyl leukotrienereceptor antagonist regulates vascular permeability by reducing vascularendothelial growth factor expression[J]. Allergy Clin Immunol, 2004,114(5): 1093-1099.]. A double-blind, random, crossed and antitheticalclinical research shows that the Montelukast can significantly suppressbronchus contraction caused by inhaling LTD4 [De Lepeleire I, Reiss T F,Rochette F, et al. Montelukast causes prolonged, potent leukotrieneD4-receptor antagonism in the airways of patients with asthma [J]. ClinPharmacol Ther, 1997, 61(1): 83.]. The Montelukast may also suppress thebronchus contraction caused by movement, Bronsky E A, et al. [Bronsky EA, Kemp J P, Zhang J, et al”. Dose related protection of exercisebronchoconstriction by montelukast, a cysteinyl leukotriene-receptorantagonist, at the end of a once-daily dosing interval [J]. ClinPharmacol Ther, 1997, 62(5):556.]. The suppression condition of theMontelukast to the bronchus contraction caused by sports is tested onthe asthma patient.

Montelukast sodium (Singulair) is the first oral leukotriene receptorantagonist. The leukotriene is one of the important mediators in aseries of inflammatory cells and inflammatory mediators in pothogenesyof the bronchial asthma, and plays a key role in generation anddevelopment of the bronchial asthma in the pathophysiology of theasthma.

Montelukast sodium (Montelukast, Singulair) is one uniquehigh-selectivity leukotriene receptor antagonist in-taken once a daycurrently, and is suitable for treating asthma of adult and child, andsyndrome of asthma and anaphylactic rhinitis asthma. Since theMontelukast has a relatively wide application range, and is convenientto intake, it has been widely recognized by clinician and used in cliniccurrently although manufacturing and application in clinic very late.

Recently, Adverse Events Reporting System (AERS) of Food and DrugAdministration (FDA) has received many reports about side effects onneuropsychiatric aspect of anti-leukotriene drugs relating to theMontelukast (Singulair) or the like, and most reports are related to theMontelukast (Singulair) that may induce a suicide tendency. The drug isthe most frequently-used anti-leukotriene prescription drug currently,and from clinical description of the suicide reports of some patients,the negative suicide events are actually caused by the drugs. Food andDrug Administration (FDA) sent an announce to medical workers andpatients on Mar. 27, 2008 [Announcement Mar. 27, 2008], and consideredthat the use of the Singulair may possible to cause changes ofbehavior/emotion and suicide tendency and behavior. The patienttransitorily does not need withdrawal, but clinical doctors shouldclosely monitor whether the patients taking these drugs present changesof behavior/emotion, and whether the patients develop suicide tendencyand behavior. For the children taking Montelukast (Singulair), it shouldbe pay attention to these children whether present appearances ofhyperactivity, attention deficit, aggressive behavior, lethargy,depression, etc.

In 2009, after investigations, FDA officially informed that three asthmadrugs including Montelukast (Singulair) have a possibility to causemental-health problems, and manufacturers of anti-leukotriene drugsshould mark drug-induced risks on drug label. FDA issued a declarationon its website to indicate that some asthma patients taking Montelukast(Singulair), Accolte, and Zileuton appear side effects of depressiveillness, anxiety, suicide tendency, etc. This FDA investigation verifiesthat three asthma drugs including Montelukast (Singulair) have apossibility to cause mental-health problems. FDA stated that bothclinical doctors and asthma patients should clearly know potential risksof these drugs to the mental health.

A spokesman from Merck Corporation which is a manufacturer ofMontelukast (Singulair) stated that the risk of neuropsychiatric sideeffects has been noted in the “negative reaction” part of theinstructions of Montelukast (Singulair), and it will be described in thepart of matters needing attention of the drug in future.

The prior art has described some quinoline-containing compounds servedas antagonist and having active effects to leukotrienes.

For example, EP318,093 (Merck) and CN1061407A (Merck Frosst Canada Inc.)describe a compound having structure A. A compound having a structuralformula B has been disclosed in WO89/12629 (Rorer) and US005565473A.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a cyclopropylunsaturated quinoline compound used as leukotriene receptor antagonistand applications thereof.

The invention employs the following technical solutions:

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

wherein:

R¹ and R² represent H, halogen, —CF₃, —CN, —NO₂, or N₃;

R³ represents lower alkyl, lower alkenyl, —CF₃, —CH₂F, —CHF₂, CH₂CF₃,substituted or unsubstituted phenyl, substituted or unsubstitutedbenzyl, substituted or unsubstituted 2-phenethyl or a ring of up to8-members containing 0 to 2 heteroatoms formed with two R² groupsconnected to one and the same carbon atom, wherein the heteroatoms areselected from O, S and N;

R⁴ represents H or R³;

R⁵ represents H or a cation corresponding to a pharmaceuticallyacceptable salt;

R⁶ and R⁷ represent H, halogen, —CF₃, —CN, —NO₂, or N₃.

The lower alkyl is C1-8 alkyl, and the lower alkenyl is C1-8 alkenyl.

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

An application of the cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist in preparing a drug for treatingand/or preventing and/or delaying and/or providing adjuvant therapy forasthma and/or allergic rhinitis and asthma syndromes.

A drug composition includes the above cyclopropyl unsaturated quinolinecompound or its pharmaceutically acceptable salts, hydrates, solvates,or prodrugs used as leukotriene receptor antagonist.

The above drug composition further includes other pharmaceuticallyexcipients.

The beneficial effects of the present invention are as follows:

The series compound of the present invention has favorable foreground inpreparing the drugs for treating and/or preventing and/or delayingand/or providing adjuvant therapy for asthma and/or allergic rhinitisand asthma syndromes.

Specifically, acute toxicity tests in KM mice shows that a series ofcyclopropyl unsaturated quinoline compound prepared according to thepresent invention is relatively safe, reliable and innoxious in atherapeutic dosage range.

In-vivo anti-asthmatic pharmacodynamics tests results shows that anisomeric compound particularly has a favorable anti-asthmatic effect onasthma guinea pig models and has more significant effects than themontelukast control group with the same dosage, which is a verypromising candidate lead compound for developing highly-effective andlow-toxicity anti-asthmatic drugs.

The series compounds of the present invention has favorable foregroundin preparing drugs for treating and/or preventing and/or delaying and/orproviding adjuvant therapy for asthma and/or allergic rhinitis andasthma syndromes.

DETAILED DESCRIPTION OF THE INVENTION

A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist has a structural formula shown below:

Preferably,

R¹ and R² represent H, halogen, —CF₃, —CN, —NO₂, or N₃;

R³ represents lower alkyl, lower alkenyl, —CF₃, —CH₂F, —CHF₂, CH₂CF₃,substituted or unsubstituted phenyl, substituted or unsubstitutedbenzyl, substituted or unsubstituted 2-phenethyl or a ring of up to8-members containing 0 to 2 heteroatoms formed with two R² groupsconnected to one and the same carbon atom, wherein the heteroatoms areselected from O, S and N;

R⁴ represents H or R¹;

R⁵ represents H or a cation corresponding to a pharmaceuticallyacceptable salt; and

R⁶ and R⁷ represent H, halogen, —CF₃, —CN, —NO₂, or N₃.

Further preferably, the lower alkyl is a C1-8 alkyl, and the loweralkenyl is a C1-8 alkenyl.

In the above compounds, the acid groups may form salts with bases, andexamples of forming salts with bases include but are not limited to:forming salts with inorganic alkali metal hydroxides, alkali metalcarbonates, alkali metal bicarbonates, alkaline earth metal hydroxides,alkaline earth metal carbonates, or alkaline earth metal bicarbonates;and forming salts with organic bases such as procaine, and forming saltswith basic amino acids such as lysine.

Preferably, the cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist is at least one of the followingcompounds:

In the above compounds, basic groups may form salts with acids, andexamples of forming salts with acids include but are not limited to:forming salts with inorganic acids, especially with haloid acids (e.g.hydrochloric acid, hydrobromic acid, and hydroiodic acid), nitric acid,sulfuric acid, phosphoric acid, carbonic acid and the like; formingsalts with lower alkyl sulfonic acids such as methane sulfonic acid andtrifluoromethane sulfonic acid; forming salts with aryl sulphonic acidssuch as benzenesulfonic acid or p-toluenesulfonic acid; forming saltswith organic acids such as an acetic acid, fumaric acid, tartaric acid,oxalic acid, citric acid, maleic acid, malic acid or succinic acid; andforming salts with amino acids such as aspartic acid or glutamic acid.

Application of the cyclopropyl unsaturated quinoline compound orpharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist in preparing drugs for treatingand/or preventing and/or delaying and/or providing adjuvant therapy forasthma and/or allergic rhinitis and asthma syndromes.

A drug composition includes the above cyclopropyl unsaturated quinolinecompounds or pharmaceutically acceptable salts, hydrates, solvates, orprodrugs used as leukotriene receptor antagonist.

The above drug composition further includes pharmaceutically excipients.

Preferably, the excipient includes at least one of the following:solvents, propellants, solubilizers, stabilizers, glidants, corrigents,preservatives, suspending agents, coating materials, aromatics,antiadherents, chelating agents, penetration enhancers, pH adjusters,buffers, plasticizers, cosolvents, emulsifiers, colorants, adhesives,disintegrating agents, filler, lubricants, wetting agents, osmolarityregulators, surfactants, foaming agents, defoaming agents, thickeners,inclusion agents, humectants, absorbents, diluents, flocculents andanticoagulants, filter aids, and releasing retardents.

The drug composition of the present invention may be prepared intovarious dosage forms: classifying according to the dispersion system ofthe dosage form, which may specifically be formulated into the followingdosage forms including solution forms, colloidal solution forms,emulsion forms, suspension forms, gas dispersion forms, particledispersion forms and solid dispersion forms; classifying according tophysical state of materials, which may specifically be formulated intothe following dosage forms including liquid forms (such as aromaticwater, solution, injection, mixture, lotion, liniment, etc), gas forms(such as aerosol, spray, etc), solid forms (such as powder, pill,tablet, membrane, etc), and semi-solid forms (such as ointment,suppositories, paste, etc); and classifying according to administrationroute, which may specifically be formulated into the following formsincluding dosage forms for gastrointestinal tract administration, anddosage forms for parenteral administration.

A synthetic route of the compound according to the present invention isas follows:

The present invention will be further described hereinafter withreference to the preferred embodiments.

Example 1: Synthesis of Fragment Compound 2

(1) Synthesis of Compound 2-2

At 0 to 5° C., a triethylamine (104 mmol) and a p-toluenesulfonyl azide(20.5 g, 104 mmol) solution in acetonitrile (100 mL) were sequentiallyadded into a diethyl malonate (87 mmol) solution in acetonitrile (150mL). After the addition was completed, the mixture was stirred at roomtemperature overnight. When the reaction was finished, the reactionsolution was concentrated under reduced pressure, and then was addedwith 100 mL ethyl ether. The organic layer was sequentially washed by 1Naqueous sodium hydroxide (50 mL), water (50 mL) and saturated brine (50mL), dried by anhydrous sodium sulfate, and then was concentrated underreduced pressure to obtain 17 g compound 2-2 (yield: 100%).

¹H NMR (CDCl₃ 400 MHz): δ4.30-4.25 (q, J=6, 4H), δ 1.31-1.27 (t, J=6,6H).

(2) Synthesis of Compound 2-3

At −60° C., 1-chloro-1-vinyl fluoride (14.9 mol) was added into amethylene chloride (20 L) suspension ofTetrakis(triphenylacetato)dirhodium(II) (48 g) and powder molecularsieve (720 g), then the reaction solution was warmed up to −35° to −40°C., and was added with the solution of the compound 2-2 (11.47 mol) inmethylene chloride (4 L). After the addition was completed, the reactionmixture was stirred at room temperature overnight. The reaction solutionwas filtered, and the filtrate was concentrated to obtain 2.9 Kg yellowoily crude compound 2-3, which was directly used in next reactionwithout further purification.

(3) Synthesis of Compound 2-4

At 80° C., raney nickel (380 g) and ethylenediamine (632 g) were addedinto the solution of crude compound 2-3 (3.35 mol) in N-methylpyrrolidone (4.5 L), which was filled with hydrogen to react overnight.After the reaction was completed, the reaction solution was cooled toroom temperature and was added with ethyl acetate (15 L), then it wasfiltered and the filtrate was sequentially washed by 2N aqueoushydrochloric acid solution and saturated brine, dried by anhydroussodium sulfate and filtered, then the filtrate was concentrated underreduced pressure, and was purified through column chromatography toobtain 518 g compound 2-4 (yield: 65%).

¹H NMR (CDC₃ 400 MHz): δ 4.75-4.48 (m, 1H), δ 4.29-4.25 (q, J=6, 4H), δ1.77-1.53 (m, 2H), δ 1.33-1.28 (t, J=6, 6H).

(4) Synthesis of Compound 2-5

At 0° C., the solution of the compound 2-4 (10.7 mmol) intetrahydrofuran (15 mL) was slowly added dropwise into the suspension ofLiAlH₄ (35.4 mmol) in tetrahydrofuran (100 mL), and then the mixture wasstirred at room temperature for 1 h. After completing the reaction,adding water to quench the reaction. The suspension was filtered throughCelite, and the filtrate was concentrated under reduced pressure, andthen purified through column chromatography to obtain 1 g compound 2-5(yield: 78%).

¹H NMR (CDC₃ 400 MHz): δ 4.70-4.54 (m, 1H), δ 4.09-2.98 (m, 6H), δ0.95-0.82 (m, 2H).

(5) Synthesis of Compound 2-6

The solution of the compound 2-5 (250 mmol) in DMF (225 mL) was addedinto a 1 L reaction flask which was equipped with a vacuum distillationunit; when the reaction temperature reached 75° C./50 torr, 25 mL DMFwas distilled out, then the solution (81.6 mL, 300 mmol) of isopropylsulfite in toluol was added into the reaction solution, wherein 50 mLtoluol could be collected at 52° C./55 torr. Then sodium tert-butoxide(2 M in THF, 2.0 mL) was added into the reaction solution, the mixturewas distilled at 35°/50 torr and 30 mL fraction could be collected; thetemperature was continuously raised up to 70° C./50 torr and the mixturewas distilled, 60 mL fraction could be collected. After that sodiumtert-butoxide (2 M in THF, 1.0 mL) was added, and the mixture wascontinuously distilled at 60-75° C./50 torr and 60 mL fraction could becollected. Then sodium tert-butoxide (2 M in THF, 0.5 mL) was added, andthe mixture was continuously distilled at 70-75° C./50 torr, thedistillation was stopped when 30 mL fraction was collected. The reactionsolution was stirred at 70° C. for 1 h, then cooled to room temperature,and was processed to obtain a compound 2-6 solution.

(6) Synthesis of Compound 2-7

Sodium cyanide (275 mmol) and sodium iodide (50 mmol) were added intothe compound 2-6 solution above, which warmed up to 70° C. and stirredfor 1 h, and then was stirred vigorously for 4h; After that toluol (400mL) was slowly added at 70° C., and then water (6 mL) was slowly addeddropwise for 30 min. After the dropwise addition was completed,distillation under a reduced pressure was performed, and 100 mL toluolcan be distilled out, then the reaction solution was cooled to 10° C.and filtered, the filter cake was rinsed with toluol (100 mL), and thefiltrate was combined and concentrated and purified through columnchromatography to obtain 24 g compound 2-7 (yield of the two steps:74%).

¹H NMR (CDCl₃ 400 MHz): δ 4.67-4.48 (m, 1H), δ 3.88-3.33 (m, 2H), δ2.76-2.34 (m, 2H), δ 1.11-0.91 (m, 2H).

(7) Synthesis of Compound 2-8

Triphenylphosphine (0.28 mol) was dissolved in 300 mL acetonitrile,cooled to −8° C., then was slowly added dropwise with bromine (0.27mol), and the reaction solution was stirred at 5° C. until yellow colordisappeared, then the solution of the compound 2-7 (0.27 mol) inacetonitrile was added dropwise and the temperature was kept below 10°C.; after the dropwise addition was completed, the reaction solution washeated to 60° C. and stirred for 15-20 min. Then the reaction solutionwas cooled to a temperature below −10% and kept for 1h, and filtered;then the filter cake was rinsed with low temperature acetonitrile (2×100mL), and the filtrate was combined and concentrated under reducedpressure, the concentrate was dissolved in methyl tertiary butyl ether(100 mL), and was stirred at −8° C. to 3° C. for more than 1h followedby filtering, and the filter cake was rinsed with low temperature methyltertiary butyl ether (2×100 mL), and the filtrate was combined andconcentrated to obtain 45 g compound 2-8 (yield: 87%).

¹H NMR (CDCl₃ 400 MHz): δ 4.69-4.50 (m, 1H), δ 3.51-3.24 (m, 2H), δ2.56-2.37 (m, 2H), δ 1.18-1.01 (m, 2H).

(8) Synthesis of Compound 2-9

The compound 2-8 (0.187 mol), acetone (165 mL) and thiourea (0.189 mol)were added into a 500 mL double-mouth bottle, the reaction solution washeated and reflowed for 12h, then the reaction solution was cooled to−8° C. to 3° C., continuously stirred for more than 1 h, and filtered;the filter cake was rinsed with low temperature acetone (2×25 mL), thenthe filter cake was pulped for 5h with 87 mL acetone, and filtered, thenthe filter cake was re-rinsed with low temperature acetone (2×25 mL),sucked dry, and dried under vacuum to obtain 44 g compound 2-9 (yield:90%).

(9) Synthesis of Fragment Compound 2

Under nitrogen protection, the compound 2-9 (0.04 mol) and 20% deaeratedsodium hydroxide solution (38.3 mL) were added into a 100 mL three-mouthbottle, heated and reflowed for 14h, and then cooled to roomtemperature, added with deaerated ethyl acetate, continuously cooled to−5 to 5° C., and then 85% formic acid was added to adjust the pH valueto between 3.5 and 4.0. The organic phase was separated and the aqueousphase was continuously extracted with ethyl acetate, then the organicphase was combined and washed with water, dried by anhydrous sodiumsulfate, and concentrated under reduced pressure to obtain a crudeproduct, and the crude product was further crystallized byn-hexane toobtain 5 g fragment compound 2 (yield: 76%).

¹H NMR (CDCl₃ 400 MHz): δ 8.87 (br, 1H), δ 4.61-4.43 (m, 1H) δ 2.91-2.66(m, 2H), δ 2.56-2.22 (m, 2H), δ 1.49-1.45 (m, 1H), 0.99-0.84 (m, 2H).

¹³C NMR (CDCl₃ 500 MHz): δ 178, δ177, δ77, δ76; δ 36, δ 33, δ 29, δ 26,δ24, δ23; δ 18, δ 16.

¹⁹F NMR (CDCl₃ 470 MHz): δ −214: δ −219.

Example 2 Synthesis of Compound 4

The synthesis scheme is as follows:

(1) Synthesis of Compound 3

(2-fluoro-cyclopropyl)-1-mercaptoethyl acetic acid (33 mmol), cesiumcarbonate (98 mmol) and polyethylene glycol-400 (10 g) were placed intoa flask, added with DMF (50 mL), stirred for 1h under room temperature.Then the reaction solution was cooled to −15° C., dropwise added withthe solution of the compound 1 (33 mL) in THF (45 mL); after thedropwise addition was completed, the reaction solution was warmed up toroom temperature and stirred for 8h. 1 M hydrochloric solution (100 mL)was added, and the mixture was extracted by ethyl acetate (3×50 mL),then the organic phase was combined and washed with saturated brine,dried and concentrated to obtain a crude product, then the crude productwas crystallized with ethyl acetate/petroleum ether to obtain 16 gyellow solid compound (yield: 80%).

¹H NMR (CDCl₃ 400 MHz): δ 8.09-8.06 (m, 2H), δ 7.75-7.48 (m, 4H), δ7.45-7.31 (m, 6H), δ 7.16-7.11 (m, 3H), δ 5.20 (br, 1H), δ 4.61-4.36 (m,1H), δ 4.01-3.98 (m, 1H), δ 3.23-3.02 (m, 1H), δ 2.94-2.51 (m, 4H), δ2.35-2.08 (m, 4H), δ 1.62-1.61 (d, 6H), δ 0.93-0.86 (m, 2H).

MS: [M+H], 605.2.

(2) Chiral Resolution of Compound 3

By chiral column separation, compound 3-1 (cis) and compound 3-2 (trans)could be obtained.

Compound 3-1 (cis):

¹H NMR (CDCl₃ 400 MHz): δ 8.08-8.04 (m, 2H), δ 7.75-7.47 (m, 4H), δ7.45-7.32 (m, 6H), δ 7.17-7.11 (m, 3H), δ 4.84 (br, 1H) δ 4.52-4.27 (m,1H), δ 4.01-3.93 (m, 1H), δ 3.19-3.14 (m, 1H), δ 2.93-2.52 (m, 4H), δ2.38-2.18 (m, 4H), δ 1.61-1.60 (d, 6H), δ 0.98-0.76 (m, 2H).

¹⁹F NMR (CDCl₃ 470 MHz): δ −216.

Compound 3-2 (trans):

¹H NMR (CDCl₃ 400 MHz): ¹H NMR (CDCl₃ 400 MHz): δ 8.07-8.04 (m, 2H), δ7.76-7.47 (m, 4H), δ 7.45-7.33 (m, 6H), δ 7.17-7.10 (m, 3H), δ 4.63 (br,1H), δ 4.74-4.36 (m, 1H), δ 4.07-3.99 (m, 1H), δ 3.24-3.13 (m, 1H), δ2.94-2.53 (m, 4H), δ 2.39-2.18 (m, 4H), δ 1.62-1.60 (d, 6H), δ 0.93-0.81(m, 2H).

¹⁹F NMR (CDCl₃ 470 MHz): δ −217.

(3) Synthesis of Compound 4

At room temperature, NaOH (8.7 mol) was firstly dissolved in methanol(30 mL), then the alkaline liquor was added into the solution of thecompound 3 (8.5 mol) in methanol (32 mL), stirred for 30 min, and thenadded with a suitable amount of activated carbon, stirred for 30 min,and filtered by paving Celite, then the filtrate was dried by rotaryevaporation, and dried under vacuum to obtain compound 4 (yield: 90%).

¹H NMR (CDC₃ 400 MHz): δ 8.03-8.00 (m, 2H), δ 7.67-7.55 (m, 4H), δ7.43-7.33 (m, 6H), δ 7.09-7.02 (m, 3H), δ 4.58-4.31 (m, 1H), δ 4.00-3.93(m, 1H), δ 3.28-3.16 (m, 1H), δ 2.86-2.53 (m, 4H), δ 2.23-2.09 (m, 4H),δ 1.59-1.55 (d, 6H), δ 0.70-0.53 (m, 2H).

MS: [M+H], 605.2.

(2) Chiral Resolution of Compound 4

The compound 4 was separated through a chiral column to obtain fourcompounds with different absolute configurations, which were illustratedas follows:

By chiral column separation, a pair of enantiomers of the cis compoundwere obtained, which were 2.4 g compound 4-1 and 2.2 g compound 4-2respectively.

Compound 4-1 (CS-0090-1):

LC-MS: 4.758 min, 96.7%; [M+H], 605.2.

SFC: 4.56 min, de value: 97.79%.

Compound 4-2 (CS-0090-2):

LC-MS: 4.758 min, 99.1%; [M+H], 605.2.

SFC: 6.36 min, de value: 100%.

¹H NMR (CDCl₃ 400 MHz): δ 8.06-8.04 (m, 2H), δ 7.69-7.57 (m, 4H), δ7.46-7.33 (m, 6H), δ 7.18-7.08 (m, 3H), δ 4.41-4.24 (m, 1H), δ 3.97-3.93(m, 1H), δ 3.23-3.16 (m, 1H), δ 2.90-2.45 (m, 4H), δ 2.24-2.15 (m, 1H),δ 1.61-1.59 (d, 6H), δ 0.93-0.68 (m, 2H).

¹³C NMR (CDCl₃ 500 MHz): δ 176.3, δ 156.8, δ148.1, δ145.2, δ 143.4,δ143.2, δ140.1, δ136.5, δ136.4, δ135.7, δ135.3, δ131.5, δ129.1, δ128.7,δ128.5, δ127.6, δ127.2, δ127.1, δ126.7, δ126.4, δ 125.7, δ125.6, δ125.4,δ119.2, δ75.4, δ73.7, δ50.3, δ 39.8, δ 36.1, δ34.5, δ32.3, δ31.8, δ31.7, δ21.4, δ 17.8.

¹⁹F NMR (CDC₃ 470 MHz): δ −215.

By chiral column separation, only 30 g racemate of the trans compound(compound 4-3 and 4-4(1:1) can not be separated by a chiral column) wereobtained.

Compound 4-4 (CS-0090-4)

LC-MS: 9.322 min, 99.1%; [M+H], 605.2.

HPLC: 8.689 min, 99.1%.

¹H NMR (CDCl₃ 400 MHz): δ 8.07-8.04 (m, 2H), δ 7.76-7.55 (m, 4H), δ7.47-7.33 (m, 6H), δ 7.17-7.10 (m, 3H), δ 4.63-4.36 (m, 1H), δ 4.06-4.01(m, 1H), δ 3.19-3.16 (m, 1H), δ 2.91-2.37 (m, 4H), δ 2.26-2.22 (m, 4H),δ 1.62-1.57 (d, 6H), δ 0.93-0.81 (m, 2H).

¹³C NMR (CDCl₃ 500 MHz): δ 174.7, δ 156.9, δ 147.8, δ 145.1, δ 143.6, δ143.4, δ 140.1, δ 136.5, δ 136.3, δ 135.8, δ 135.7, δ 131.5, δ 129.1, δ128.7, δ 128.6, δ 127.4, δ 127.7, δ 127.1, δ 126.7, δ 126.4, δ 125.6, δ125.5, δ 125.4, δ 119.1, δ76.7, δ 73.8, δ50.5, δ 39.9, δ 38.1, δ32.8,δ32.2, δ31.8, δ31.6, δ21.9, δ 18.4.

¹⁹F NMR(CDCl₃ 470 MHz): δ −216.

Test Parts:

Anti-Asthmatic Pharmacodynamics Experiment of Leukotriene ReceptorAntagonist CS-0090 Series Compounds

Part One: Acute Toxicity Experiment in Mice

1 Instruments and Materials

1.1 Experimental Animals

1.1.1 Strain and level: KM mice, ordinary level.

1.1.2 Number and sex: 40, half male and half female.

1.1.3 Weight: the mice were 18 to 22 g at purchase.

1.1.4 Supply Organization: provided by Laboratory Animal Center ofGuangzhou University of Chinese Medicine.

1.1.5 Recognition method: hair staining method was adopted. The micewere numbered by saturated picric acid, and the hair on different partsof the mouse body surface was spotted to represent different numbers.The mice were marked and recognized by both numbers of the mouse skinstaining and cages.

1.1.6 Breeding and management: the animals were raised in a conventionalanimal room, and the experimental animal license number was: SCXK(Guangdong) 2013-0020. Animal breeding condition: 10 mice/cage and groupbreeding, breeding temperature and humidity: 20 to 25° C., 40 to 70%;With 10h:14h day and night intermittent lighting, the animals werefreely access to food and water during the experiment. Conditions in thebreeding room were kept stable all the time to ensure the reliability ofthe experimental results.

1.1.7 Quarantine: the mice purchased were quarantined for seven days.During this period, the animals were checked once a day. If anyunhealthy animal was found, eliminated immediately, and healthy animalswere selected for the experiment.

1.2 Main Reagents

anhydrousethanol (Tianjin Damao Chemical Reagent Factory), Tween-80(Jiangsu Hai'an Petroleum Chemical Factory), deionized water(Laboratory-prepared by the College of Pharmacy of GuangdongPharmaceutical University (

)), picric acid (Beijing Xinding Pengfei Science and TechnologyDevelopment Co., Ltd. (

)), formaldehyde solution (Tianjin Damao Chemical Reagent Factory),physiological saline (Laboratory-prepared by the College of Pharmacy ofGuangdong Pharmaceutical University), Montelukast (Chem-Stone(Guangzhou) Co., Ltd. (

)), CS-0090-1, CS-0090-2 and CS-0090-4 (provided by Chem-Stone(Guangzhou) Co., Ltd., Lot No.: 20141012).

1.3 Main Instruments and Materials

0.01 mg electronic balance (Shimadzu, Japan, Product No: AUW120D),electronic balance (Shimadzu, Japan, Product No: AUY120), V7 fast mixer(Essenscien, US), electrothermal thermostatic water tank (ShanghaiYiheng Instruments Co., Ltd. (

), Product No: DK-8D), and 1 mL disposable sterile injector (Becton,Dickinson and Company, US).

2 Experimental Method

2.1 Dose Design and Grouping

2.1.1 Dose Design

2.1.1.1 Montelukast: a recommended intake dosage of montelukast forhuman is 10 mg/d (calculated by a standard human body weight of 60 kg),thereby an equivalent dosage of the mice is 1.52 mg·kg⁻¹·d⁻¹, and theadministration dosage is 1500 times of the equivalent dosage, which is2280·kg⁻¹·d⁻¹. The specific dosing regimen was: 760 mg·kg⁻¹, 0.01 ml/g,and three times a day.

2.1.1.2 CS-0090-1, CS-0090-2 and CS-0090-4: to facilitate the resultscomparison, the dosing regimens of CS-0090-1, CS-0090-2 and CS-0090-4were set to be the same as montelukast.

2.1.2 Animal Grouping

The animals were randomly divided into four groups by weights after theadaption period, i.e. a positive drug male control group, a positivedrug female control group, a CS-0090-1 male experimental group and aCS-0090-1 female experimental group, and 10 mice in each group.

2.2 Acute Toxicity Effects of CS-0090-1, CS-0090-2 and CS-0090-4 in KMMice

2.2.1 Drug Formulation Method

2.2.1.1 Montelukast: a certain amount of montelukast was weighed, anappropriate amount of Tween-80 was added to obtain a concentration of0.2% thereof and an appropriate amount of ethanol was added to obtain aconcentration of 10% thereof, and then the mixture was vortexed andcompletely dissolved, then an appropriate amount of deionized water wasadded to formulate a ready-to-use medical liquid having a concentrationof 76 mg/mL.

2.2.1.2 CS-0090-1, CS-0090-2 and CS-0090-4: a certain amount ofCS-0090-1, CS-0090-2 and CS-0090-4 were weighed, an appropriate amountof Tween-80 was added to obtain a concentration of 0.2% thereof and anappropriate amount of anhydrous ethanol was added to obtain aconcentration of 10% thereof, and then the mixture was vortexed andcompletely dissolved, then an appropriate amount of deionized water wasadded to formulate a ready-to-use medical liquid having a concentrationof 76 mg/mL.

2.2.2 Test Procedures

The animals were randomly divided into four groups by body weight afterfinishing the adaption period, i.e. a positive drug male control group,a positive drug female control group, a test reagent male experimentalgroup and a test reagent female experimental group, and 10 mice/group.The mice in each group were fasted for 12 hours before administrationwith water ad libitum. The animals in each group were administeredcorresponding therapy drugs of 0.01 ml/g of their body weight by oralgavage, three times/day, and totally administered for one day. On Day 8,the mice were dissected with their lungs and bronchi taken to be fixedand saved in a formaldehyde solution.

2.2.3 Detection Indicators

2.2.3.1 Daily observation: in the first 30 min after administration,each animal should be observed at least once; in the following 24h afteradministration, the animals should be observed regularly (time intervalof the observation was determined by the toxic reaction, time of onsetand time of recovery cycle, and particular attention should be paidwithin 4h after administration); hereinafter, the animals were observedonce a day, and were totally observed for 7 days. The administrationprocedure and the signs of toxicity and death of the animals within theobservation period were observed and recorded.

2.2.3.2 Observation of persistent toxic symptom: Additional observationswill be necessary if the animals continue to display signs of toxicity.The observations should include changes in skin and fur, eyes and mucousmembranes, respiratory, circulatory, autonomic nucleuses and centralnervous systems, somatomotor activities and behavioral patterns.Attentions should be directed to observations of tremors, convulsions,salivation, diarrhea, lethargy and coma. Observation items for the toxicmanifestions of rodent animals were showed in Table 1.

TABLE 1 Observation Items for the Toxic Manifestions of Rodent AnimalsOrgan system Observation and inspection item General toxic manifestionsCentral nervous Behavior Postures changed, abnormal sound, disturbed ordull system and somatic Action Tremor, ataxy, paralysis, eclampsia,forced action movement Reaction on various stimulations Excitable,hypergnosia, or lack of perception Cerebral and spinal reflex Weakenedor disappeared Muscle tone Tetanic, and tardive Autonomic nerves Pupilsize miosis or mydriasis system Secretion Salivation, lacrimationRespiration Nostril nasal discharge property Respiration property andrate Bradypnoea, breathing difficult, and cheyne-stokes respirationCardiovascular Cardiac palpation Bradyrhythmia, anisorhythmia, toostrong or too system weak heartbeat Gastrointestinal Belly shapeFlatuous or contracted, diarrheal or belly-bound system Feces hardnessand color Unshapen feces in black or grey Genitourinary Vulvae, mammarygland Swelled system Priapus Prolapsed Perineum Squalid Skin and furColor, and tension red, corrugated, relaxed, tetter IntegrityPiloerection Mucous Mucous membranes Mucus discharged, hyperemia,hemorrhagic membranes cyanosis cyanosis, and pallored Oral cavityUlcerous Eyes Palpebra Upper eyelid cambered Eyebulb Ophthalmoptosis ornystagmus Transparency Dimness Others Cecum or skin temperature Reducedor increased General situations Abnormal postures, and wizened

2.2.3.3 Dissection: The animals died within the observation period weresubjected to necropsy; At the end of the observation period, thesurviving mice were sacrificed by cervical dislocation for necropsy. Thepathological changes of lung and bronchus of each animal were recorded,and histopathological examination was conducted on the observed visceralorgans.

3 Results

The dosage of the mice was converted and obtained according to arecommended dosage (10 mg/d) of a positive drug montelukast for human,and used as an initial dosage (1.52 mg·kg⁻¹) of the test drugsCS-0090-1, CS-0090-2 and CS-0090-4; From a preliminary experiment, it isknown that the mice in the experimental group were not 100% dead whenadministrated a dosage of 500 times (760 mg·kg⁻¹) of the initial dosageby intragastric administration. As the CS-0090-1, CS-0090-2 andCS-0090-4 have a very low solubility in water, the dosage form can onlybe suspension; if increasing the drug concentration further, it isdifficult to form a more stable system with the drug and solvents, whichmay lead inaccurate intragastric administration dosage, which means thatthe LD50 of the CS-0090-1, CS-0090-2 and CS-0090-4 cannot be determined;therefore, only the maximum drug dosage of the CS-0090-1, CS-0090-2 andCS-0090-4 in the mice can be determined.

Acute toxicity effect tests of montelukast (which was provided byChem-Stone (Guangzhou) Co., Ltd.) in KM mice showed that none of themice died except one of the positive drug female group died in the nextday and these mice were normal in ingestion, activity behavior, urineand defecation, and had no special secretions, no toxic reactions ofnervous system, such as reduced activity, keeping still and lessmovement, alarmed drunk-like walking and other symptoms; on Day 8, ananatomical observation was performed on the mice and found that both thelungs and bronchi of the mice were normal. In view of individualdifferences, the maximum dosage of montelukast can be deemed as 760mg·kg⁻¹*3=2280 mg·kg⁻¹.

Acute toxicity effect tests of CS-0090-1, CS-0090-2 and CS-0090-4 (whichwere provided by Chem-Stone (Guangzhou) Co., Ltd.) in KM mice showedthat none of the mice died, and these mice were normal in ingestion,activity behavior, urine and defecation, and had no special secretions,no toxic reactions of nervous system, such as reduced activity, keepingstill and less movement, alarmed drunk-like walking and other symptoms;on Day 8, an anatomical observation was performed on the mice and foundthat both the lungs and bronchi of the mice were normal. Therefore, themaximum dosages of the CS-0090-1, CS-0090-2 and CS-0090-4 can also bedeemed as 760 mg·kg⁻¹*3=2280 mg·kg⁻¹, 763 mg·kg⁻¹*3=2289 mg·kg⁻¹, 754mg·kg⁻¹*3=2262 mg·kg⁻¹, respectively, which demonstrated that theCS-0090-1, CS-0090-2 and CS-0090-4 were relatively safe, reliable andinnoxious in a therapeutic dosage range.

Part Two: In-Vivo Anti-Asthmatic Pharmacodynamics Experiment of CompoundCS-0090-1

1 Instruments and Materials

1.1 Experimental Animals

1.1.1 Strain and level: guinea pigs, ordinary level.

1.1.2 Numbers and sex: 24, male.

1.1.3 Weight: the guinea pigs were 180 to 220 g at purchase.

1.1.4 Supply Organization: provided by Laboratory Animal Center ofGuangzhou University of Chinese Medicine.

1.1.5 Recognition method: hair staining method was adopted. The guineapigs were numbered by saturated picric acid, and the hair on differentparts of the guinea pigs body surface was spotted to represent differentnumbers. The guinea pigs were marked and recognized by both numbers ofthe guinea pigs skin staining and cages.

1.1.6 Breeding and management: the animals were raised in a conventionalanimal room, and the experimental animal license number was: SCXK(Guangdong) 2013-0020. Animal breeding condition: 6 guinea pigs/cage andgroup breeding, breeding temperature and humidity: 20 to 25° C., 40 to70%; With 10h:14h day and night intermittent lighting, and the animalswere freely access to food and drink water during the experiment.Conditions in the breeding room were kept stable all the time to ensurethe reliability of the experimental results.

1.1.7 Quarantine: the guinea pigs purchased were quarantined for threedays. During this period, the animals were checked once a day. If anyunhealthy animal was found, eliminated immediately, and healthy animalswere selected for the experiment.

1.2 Main Reagents

acetylcholine chloride (Shanghai SSS Reagent Co., Ltd.), histamine(Beijing Lvyuan Bode Biological Technology Co., Ltd. (

)), anhydrous ethanol (Tianjin Damao Chemical Reagent Factory), Tween-80(Jiangsu Hai'an Petroleum Chemical Factory), picric acid (BeijingXinding Pengfei Science and Technology Development Co., Ltd.), deionizedwater (Laboratory-prepared by the College of Pharmacy of GuangdongPharmaceutical University), Montelukast (Chem-Stone (Guangzhou) Co.,Ltd.), and CS-0090-1 (Chem-Stone (Guangzhou) Co., Ltd.).

1.3 Main Instruments and Material s

V7 fast mixer (Essenscien, US), electrothermal thermostatic water tank(Shanghai Yiheng Instruments Co, Ltd., Product No: DK-8D), Yuwell 402AIultrasonic atomizer (Jiangsu Yuyue Medical Equipment & Supply Co.,Ltd.), electronic balance (Shimadzu, Japan, Product No: AUY120), 0.01 mgelectronic balance (Shimadzu, Japan, Product No: AUW120D), and 1 mLdisposable sterile injector (Becton, Dickinson and Company, US).

2 Experimental Method

2.1 Establishment of Guinea Pig Asthma Models

Mixing-atomization induced asthma method of 0.1% histamine and 2%acetylcholine solution was adopted. 180 to 220 g male guinea pigs weretaken and placed in a lab-prepared closed container (2 L) the day beforeexperiment, inhaling a mixed solution of 0.1% histamine and 2%acetylcholine by ultrasonic atomization, a maximum atomization volumewas adjusted and pulverization was continued until the guinea pigs hadsigns of cough. Observing the time period from the beginning of theguinea pigs' inhalation to develop convulsions and tumble (which is theasthma-induced latent period). The guinea pigs having an asthma-inducedlatent period within 120s were deemed to be qualified sensitive animals,and the qualified animals were selected for grouping and administration.

2.2 Dose Design and Grouping

2.2.1 Dose Design

2.2.1.1 Montelukast: A recommended intake dosage of montelukast forhuman is 10 mg/d (calculated by a standard human body weight of 60 kg),thereby an equivalent dosage of the guinea pig is 1 mg·kg−1·d−1, and theadministration dosage is three times of the equivalent dosage, which is3 mg·kg⁻¹·d⁻¹.

2.2.1.2 CS-0090-1: one times of the equivalent dosage of montelukast wasused as a low dosage of CS-0090-1, i.e., 1 mg·kg⁻¹·d⁻¹; three times ofthe equivalent dosage of montelukast, i.e., 3 mg·kg⁻¹·d⁻¹, was used as amedian dosage of CS-0090-1, and nine times of the equivalent dosage ofmontelukast, i.e., 9 mg·kg⁻¹·d⁻¹, was used as high dosage of theCS-0090-1.

2.2.2 Animal Grouping

The guinea pigs with qualified induced asthma latent period wererandomly divided into four groups by weight, i.e., a montelukast controlgroup, a CS-0090-1 low-dosage experimental group, a CS-0090-1median-dosage experimental group, and a CS-0090-1 high-dosageexperimental group, and six guinea pigs in each group.

2.3 Anti-Asthmatic Effect of CS-0090-1 on the Guinea Pig Asthma Models

2.3.1 Drug Formulation Method

2.3.1.1 Montelukast: a certain amount of montelukast was weighed, anappropriate amount of Tween-80 was added to obtain a concentration of0.2% thereof and an appropriate amount of ethanol was added to obtain aconcentration of 10% thereof, and then the mixture was vortexed andcompletely dissolved, then an appropriate amount of deionized water wasadded to formulate a ready-to-use medical liquid having a concentrationof 0.6 mg/mL.

2.3.1.2 CS-0090-1: a certain amount of CS-0090-1 was weighed, anappropriate amount of Tween-80 was added to obtain a concentration of0.2% thereof and an appropriate amount of ethanol was added to obtain aconcentration of 10% thereof, and then the mixture was vortexed andcompletely dissolved, then an appropriate amount of deionized water wasadded to formulate a high-dosage solution having a concentration of 0.2mg/mL. A median-dosage solution having a concentration of 0.6 mg/mL, anda low-dosage solution having a concentration of 1.8 mg/mL were fomulatedby the same method, wherein the solutions were prepared ready-to-use.

2.3.1.3 A mixed solution of 0.1% histamine and 2% acetylcholine: acertain amount of histamine was weighed, and an appropriate amount ofdeionized water was added to formulate a histamine solution having aconcentration of 0.1%; a certain amount of acetylcholine was weighed,and an appropriate amount of deionized water was added to formulate aacetylcholine solution having a concentration of 2%; then the above twosolutions were mixed isometrically, wherein the solution was preparedready-to-use.

2.3.2 Test Procedures

24 guinea pigs with qualified induced asthma latent period were randomlydivided into four groups by weight, i.e., a montelukast control group, aCS-0090-1 low-dosage experimental group, a CS-0090-1 median-dosageexperimental group, and a CS-0090-1 high-dosage experimental group, andsix guinea pigs in each group. The guinea pigs in each group were fastedfor 12 hours before administration with water ad libitum, and wereadministered corresponding therapy drugs of 1 ml/100 g of their bodyweight by oral gavage once a day, and the drug was continuouslyadministered for seven days. After 1h of the last administration on theseventh day, the guinea pigs were placed in a lab-prepared closedcontainer (2 L) again to induce asthma under the same condition ofpreparing the asthma models, and observing and recording the inducedasthma latent period after the administration of the guinea pigs,wherein the latent period exceeding 6 min were calculated as 6 min.

2.3.3 Statistical Analysis

Statistical analysis was performed in Excel. The data of allexperimental results was expressed as mean±standard deviation (x±s). Byusing T-test, when P≤0.05, the two data sets had significantdifferences; and when P>0.05, the two data sets had no significantdifferences.

3 Results

No blank control group was designed in this experiment, and aself-control method before and after administration was adopted, whereinthe numbers of the animals satisfied the statistics requirements, andeach group included six guinea pigs that were successfully induced. Asshown in the Table below, when comparing the induced asthma latentperiod of the CS-0090-1 median-dosage group after administration and thesame group before administration, P<0.05, which indicated there weresignificant differences; the CS-0090-1 median-dosage significantlyprolonged the induced asthma latent period of the guinea pig, whichindicating a more noticeable effect than the montelukast control groupof the same dosage; however, the P value of the CS-0090-1 low-dosagegroup and the CS-0090-1 high-dosage group were more than 0.05, whichindicated no significant differences when compared with the inducedasthma latent period of the group theirselves before administration,which means that anti-asthmatic effects of the CS-0090-1 low-dosagegroup and the CS-0090-1 high-dosage group were not noticeable on theguinea pig asthma models, the reason may be that the low-dosageCS-0090-1 did not reach the minimum anti-asthmatic effective dosage,thus the anti-asthmatic effect on the guinea pig asthma models was notobvious. While the the median-dosage CS-0090-1 had reached the maximumeffective dosage, thus the anti-asthmatic effect on the guinea pigasthma models was not obvious when the dosage was increasedcontinuously.

TABLE 2 Result of CS-0090-1 Anti-asthmatic Effect on Guinea Pig AsthmaModels Animal Induced asthma Number Dosage latent period before Inducedasthma latent period Group (piece) (mg · kg⁻¹) administration (s) afteradministration (s) Montelukast control group 6 3 48.17 ± 4.62  63.00 ±7.13* CS-0090-1 low-dosage 6 1 49.17 ± 8.70  60.33 ± 17.11 experimentalgroup CS-0090-1 median-dosage 6 3 60.17 ± 9.47 209.83 ± 164.74*^(#)experimental group CS-0090-1 high-dosage 6 9 63.50 ± 11.57 215.67 ±78.09*^(#) experimental group Note: *compared with the induced asthmalatent period of the group itself before administration, P < 0.05, therewas significant difference; #compared with the induced asthma latentperiod of the montelukast positive control, P < 0.05, there wassignificant difference.

4 Brief Summary

In the in-vivo pharmacodynamics experiments, while compared with thepositive control drug montelukast, both the median-dosage andhigh-dosage of CS-0090-1 can significantly prolong the induced asthmalatent period of the guinea pigs (P values were both smaller than0.05),which indicates that it has a favourable anti-asthmatic effect onthe guinea pig asthma models, and is a candidate lead compound having adruggability prospect. Pharmaceutics studies may be performed to improvethe bioavailability of CS-0090-1, which could provide more experimentalbasis for further druggability evaluation.

Part Three: Studies on In-Vivo Anti-Asthmatic Pharmacodynamics ofCompound CS-0090-2

1 Instruments and Materials

1.1 Drugs and Reagents

Both montelukast and the tested drugs CS-0090-2 were provided byChem-Stone (Guangzhou) Co., Ltd.; acetylcholine chloride (acetylcholine,Shanghai SSS Reagent Co., Ltd); histamine phosphate (histamine, BeijingLvyuan Bode Biological Technology Co., Ltd.); anhydrous ethanol (TianjinDamao Chemical Reagent Factory), Tween-80 (Jiangsu Hai'an PetroleumChemical Factory); Sodium chloride (NaCl, Guangdong Guanghua Sci-TechCo., Ltd); potassium chloride (KCl, Tianjin Baishi Chemical Co., Ltd.);calcium chloride (CaCl₂, Tianjin Zhiyuan Chemical Reagent Co., Ltd. (

)); sodium hydrogen carbonate (NaHCO₃, Tianjin Zhiyuan Chemical ReagentCo., Ltd.); potassium dihydrogen phosphate (KH₂PO₄, Tianjin FuchenChemical Reagents Factory); magnesium sulfate (MgSO₃, Tianjin DamaoChemical Reagent Factory); glucose (Glucose Tianjin Baishi Chemical Co.,Ltd.); formaldehyde (Tianjin Damao Chemical Reagent Factory);physiological saline (Laboratory-prepared by the College of Pharmacy ofGuangdong Pharmaceutical University); and picric acid (Beijing XindingPengfei Science and Technology Development Co., Ltd.).

1.2 Experimental Animals

Guinea pig: 180 to 220 g of body weight (induced asthma), healthy andmale.

White mouse: Kunming strain, 20 to 25 g of body weight, half female andhalf male.

All the above animals were provided by the Lab of the College ofPharmacy of Guangdong Pharmaceutical University, and the certificationnumber of the mice was: SCXK (Guangdong) 2013-0020; and the use licensenumber of the guinea pigs was: SCXK (Guangdong) 2013-0020.

1.3 Experimental Instruments

Yuwell 402AI ultrasonic atomizer (Jiangsu Yuyue Medical Equipment &Supply Co., Ltd.); ZC⁻¹0 intelligent super thermostatic water tank(Ningbo Tianheng Instrument Factory); Xinhang JZ100 tonotransducer(Beijing Xinhang Xingye Technology Trade Co., Ltd.); MedLab biosignalacquisition and processing system (Nanjing Medease Science andTechnology Co., Ltd.); 0.1 mg electronic balance (Shimadzu, Japan,Product No.: AUY120); 0.01 mg electronic balance (Shimadzu, Japan,Product No.: AUW120D); electrothermal thermostatic water tank (ShanghaiYiheng Instruments Co., Ltd., DK-8D); BIOLAB swirl blender MB (ShanghaiBIOLAB Equipment Co., Ltd.); and ultrasonic cleaner (Dongguan KeqiaoUltrasonics Facilities Co., Ltd. (

)).

2 Experimental Method

2.1 Establishment of Histamine and Acetylcholine Induced Asthma AnimalModels of Guinea Pigs

2.1.1 Dosage Setting

The clinic dosage of montelukast was 10 mg/d, and administered once aday. A standard human body weight was set as 60 kg, then the clinicdosage was 0.17 mg·kg⁻¹. According to a equivalent dosage ratiocalculated by the body surface area of human and the guinea pig, thedosage of the guinea pig was set as 3 mg·kg⁻¹. Similar to the abovedescription, to facilitate to compare the medical effects, a high,median and low dosage of the tested drug CS-0090-2 were 9 mg·kg⁻¹, 3mg·kg⁻¹ and 1 mg·kg⁻¹ respectively.

2.1.2 Model Preparation and Dosing Scheme

180 to 220 g health guinea pigs were taken, and placed in a closed glassbell jar one by one firstly; after the guinea pigs became quiet, anatomization apparatus was started to spray an asthma-induced medicalliquid (a solution of 2% acetylcholine chloride and 0.1% histaminephosphate mixed in equal volume) for 15s, then the pulverization wasstopped, and the asthma latent period (time period from the beginning ofpulverization to onset of asthma and dyspnea, till convulsions andtumble) was observed, and qualified animals having a asthma latentperiod ≤120s were screened. The qualified guinea pigs screened wererandomly divided into four groups, which were a high-dosage group, amedian-dosage group, a low-dosage group, and a positive control grouprespectively.

On the next day, 42 qualified guinea pigs were selected, randomlydivided into four groups with 6 guinea pigs in each group, which were 3mg·kg⁻¹ of montelukast group, and 1, 3 and 9 mg·kg⁻¹ of the tested drugCS-0090-2 groups. The guinea pigs in each group were subjected to oralgavage administration continuously for 7 days with a dosage of 1 ml/100g. The guinea pigs were placed in the closed glass bell jar one by onein 1 h after the administration on the last day, and pulverization wasperformed for inducing asthma under the same experimental conditions tothe screen experiment. The asthma latent period and the number of theconvulsions animals were recorded, wherein the animals did not tumblebeyond 360s were calculated as 360s.

2.2 Statistical Test

Experimental data was represented by ±s, and data statistics wasprocessed by SPSS11.0.

The asthma latent period was prolonged significantly (P<0.05). After theCS-0090-2 (1, 3 and 9 mg·kg⁻¹) was administered for seven days, theasthma latent period of the guinea pig was not changed apparentlythrough self-control before and after administration.

TABLE 3 Anti-asthmatic Effect of CS-0090-2 on Histamine andAcetylcholine Induced Asthma Guinea Pigs (x ± s) Induced asthma latentperiod (s) Animal within 6 min Number Dosage Before After Group (piece)(mg · kg⁻¹) administration administration Montelukast 6 3 48.2 ± 4.659.8 ± 7.1* CS-0090-2 6 1 47.8 ± 7.6 126.3 ± 155.9 low-dosage CS-0090-26 3 54.8 ± 7.5 132.3 ± 151.9 median-dosage CS-0090-2 6 9  60.7 ± 10.157.0 ± 6.1  high-dosage Note: compared with the situation beforeadministration: *P < 0.05; **P < 0.01.

4 Brief Summary

In the induced asthma experiment of guinea pigs, the novel compoundCS-0090-2 had no antagonism effects on the asthma of the guinea pigs.

Part Four: Studies on In-Vivo Anti-Asthmatic Pharmacodynamics ofCompound CS-0090-4

1 Instruments and Materials

1.1 Experimental Animals

Healthy and male guinea pigs with a body weight of 180 to 220 g(provided by the Laboratory Animal Center of Guangdong PharmaceuticalUniversity, and the certification number was SCXK (Guangdong)2013-0020).

1.2 Main Instruments

0.01 mg electronic balance (Shimadzu, Japan, Product No.: AUY120),electronic balance (Shimadzu, Japan, Product No.: AUY120), V7 fast mixer(Essenscien, US), Yuwell 402AI ultrasonic atomizer (Jiangsu YuyueMedical Equipment & Supply Co., Ltd.); and electronic balance(SHIMADZU/Shimadzu, Product No.: AUW120D)

1.3 Main Reagents and Materials

acetylcholine chloride (Shanghai SSS Reagent Co., Ltd., lot number:20021018), histamine phosphate (Shanghai Lizhu Dongfang Biotechnics Co.,Ltd. (

), lot number: 010310), montelukast (provided by Chem-Stone (Guangzhou)Co., Ltd.), and CS-0090-4 (provided by Chem-Stone (Guangzhou) Co.,Ltd.).

2 Experimental Method

2.1 Formulation of Asthma-Induced Solution

0.2 g choline and 0.01 g histamine were weighed and put into beakersrespectively, and then respectively each beaker were added with 10 mldistilled water and blended to homogeneity.

2.2 Influences of CS-0090-4 on the Asthma Latent Period of Guinea Pigs

2.2.1 Establishment of Induced Asthma Animal Models of Guinea Pigs

The purchased guinea pigs were quarantined for 3 days. During thisperiod, the animals were checked once a day, any unhealthy animal foundshould be eliminated immediately, and healthy animals were selected forthe experiment. The healthy guinea pigs with body weight of 180 to 220 gwere taken and placed in a closed glass bell jar one by one firstly;after the guinea pigs became quiet, an atomization apparatus was startedto spray an asthma-induced medical liquid (a solution of 2%acetylcholine chloride and 0.1% histamine phosphate mixed in equalvolume) until the guinea pigs tumbled, then the pulverization wasstopped, and the asthma latent period (time period from the beginning ofpulverization to onset of asthma and dyspnea, till convulsions andtumble) was observed, and qualified animals having a asthma latentperiod ≤120s were screened.

2.2.2 Dosage Settings

Literatures reported that a recommended intake dosage of montelukast fora human body is 10 mg/d (calculated by a standard human body weight of60 kg), and a daily dosage was 0.17 mg·kg⁻¹. According to a multipleprofile to convert the adult dosage into an animal dosage, it could beknown that the CS-0090-4 dosages of the guinea pigs were low-dosage (1mg·kg⁻¹), median-dosage (3 mg·kg⁻¹) and high-dosage (9 mg·kg⁻¹), and thedosage of montelukast of the positive control group was median-dosage (3mg·kg⁻¹).

2.2.3 Dosing Scheme

The qualified guinea pigs screened were randomly divided into fourgroups, which were a high-dosage group, a median-dosage group, alow-dosage group, and a positive control group. The guinea pigs in eachgroup were subjected to oral gavage administration continuously for 7days with a dosage of 1 ml/100 g. The induced asthma latent period wasdetermined in 1h after administration on the last day.

3 Results

3.1 Influences of CS-0090-4 on the Asthma Latent Period of Guinea Pigs

The histamine and the acetylcholine inhaled in by the guinea pigs wereacted on H1 and M receptors of airway epithelial cells of the guineapigs, which made the smooth muscle of the airway contracted, and madethe guinea pigs difficult to breathe, wherein this model could simulatethe airway obstruction symptom when the asthma attacks. From Table 4, itcan be seen that significant differences (P<0.05) present between thelatent periods before and after the administration of the 9 mg·kg⁻¹group in the CS-0090-4 dosage groups, and there were no significantdifferences (P>0.05) between the latent periods before and after theadministration of the 1 mg·kg⁻¹ group and the 3 mg·kg⁻¹ group in theCS-0090-4 dosage groups, which indicated that CS-0090-4 can remarkablyprolong the asthma latent period with the dosage of 9 mg·kg⁻¹, while theasthma latent period can not be remarkably prolonged with the dosages of1 mg·kg⁻¹ and 3 mg·kg⁻¹.

3.2 Influences of Montelukast on the Asthma Latent Period of Guinea Pigs

From Table 3, it can be seen that there were significant differences(P<0.05) between the latent periods before and after the administrationof the 3 mg·kg⁻¹ of montelukast, which indicated that the asthma latentperiod can be remarkably prolonged with the dosage of 3 mg·kg−1 ofmontelukast.

TABLE 4 Anti-asthmatic Effect of CS-0090-4 on Histamine andAcetylcholine Induced Asthma Guinea Pigs (x ± s) Asthma Dosage Asthmalatent period latent period after Group (mg · kg⁻¹) beforeadministration administration Montelukast 3 48.17 ± 4.62* 59.83 ± 7.08 149.67 ± 9.27  52.83 ± 2.93 CS-0090-4 3 59.50 ± 15.62 60.17 ± 7.33 945.17 ± 4.49* 53.33 ± 6.38 Compared with the induced asthma latentperiod before administration, *P < 0.05.

4. Brief Summary

In the in-vivo anti-asthmatic experiment of guinea pigs, the CS-0090-4had significant anti-asthmatic effect on the acetylcholine-histamineinduced guinea pig asthma with a dosage of 9 mg·kg⁻¹, which canremarkably prolong the asthma latent period of the guinea pigs, whilethe montelukast had significant anti-asthmatic effect (P<0.05) on theacetylcholine-histamine induced guinea pig asthma with a dosage of 3mg·kg⁻¹. Accordingly, the anti-asthmatic effect of CS-0090-4 is lessmarked than montelukast.

CONCLUSIONS

1. The acute toxicity tests results indicated that: the acute toxicaleffect experiment of the CS-0090-1, CS-0090-2 and CS-0090-4 (which wereprovided by Chem-Stone (Guangzhou) Co., Ltd.) on KM mice showed thatnone of the mice died, and these mice were normal in ingestion, activitybehavior, urine and defecation, and had no special secretions, no toxicreactions of the nervous system, such as reduced activity, keeping stilland less movement, alarmed drunk-like walking and other symptoms; on Day8, an anatomical observation was performed on the mice and found thatboth the lungs and bronchi of the mice were normal. Therefore, themaximum dosages of the CS-0090-1, the CS-0090-2 and the CS-0090-4 canalso be deemed as 2280 mg·kg⁻¹, 2289 mg·kg⁻¹ and 2262 mg·kg⁻¹respectively, which demonstrates that the CS-0090-1, the CS-0090-2 andthe CS-0090-4 are relatively safe, reliable and innoxious in atherapeutic dosage range.

2. The in-vivo anti-asthmatic pharmacodynamics tests results ofCS-0090-1 showed that: the high-dosage CS-0090-1 significantly prolongedthe induced asthma latent period of the guinea pigs, which indicatesthat it has good anti-asthmatic effect on the guinea pig asthma modelsand its effect is more significant than the montelukast control groupwith the same dosage (P<0.05), and is a very promising candidate leadcompound for developing highly-effective low-toxicity anti-asthmaticdrugs. Based on this, pharmaceutics studies may be performed to improvethe bioavailability of CS-0090-1, which could provide more experimentalbasis for further druggability evaluation.

3. The in-vivo anti-asthmatic pharmacodynamics tests results ofCS-0090-2 showed that: the compound CS-0090-2 had no antagonism effecton the asthma of the guinea pigs.

4. The in-vivo anti-asthmatic pharmacodynamics tests results ofCS-0090-4 showed that: in the in-vivo anti-asthmatic experiment ofguinea pigs, the CS-0090-4 had significant anti-asthmatic effect on theacetylcholine-histamine induced guinea pig asthma under a high dosage (9mg·kg⁻¹) administration condition, which could remarkably prolong theasthma latent period of the guinea pigs. While the montelukast had asignificant anti-asthmatic effect on the acetylcholine-histamine inducedguinea pig asthma under a dosage of 3 mg·kg⁻¹, which could remarkablyprolong the asthma latent period of the guinea pigs. Accordingly, theasthma treatment effect of the CS-0090-4 was lower than the positivecontrol drug with respect to the positive control drug montelukast.

1. A cyclopropyl unsaturated quinoline compound or its pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist, having a structural formula as follows:

wherein: R¹ and R² represent H, halogen, —CF₃, —CN, —NO₂, or N₃; R³represents lower alkyl, lower alkenyl, —CF₃, —CH₂F, —CHF₂, CH₂CF₃,substituted or unsubstituted phenyl, substituted or unsubstitutedbenzyl, substituted or unsubstituted 2-phenethyl or a ring of up to8-members containing 0 to 2 heteroatoms formed with two R² groupsconnected to one and the same carbon atom, wherein the heteroatoms areselected from O, S and N; R⁴ represents H or R³; R⁵ represents H or acation corresponding to a pharmaceutically acceptable salt; R⁶ and R⁷represent H, halogen, —CF₃, —CN, —NO₂, or N₃.
 2. The cyclopropylunsaturated quinoline compound or its pharmaceutically acceptable salts,hydrates, solvates, or prodrugs used as leukotriene receptor antagonistaccording to claim 1, characterized in that the lower alkyl is C1-8alkyl, and the lower alkenyl is C1-8 alkenyl.
 3. The cyclopropylunsaturated quinoline compound or its pharmaceutically acceptable salts,hydrates, solvates, or prodrugs used as leukotriene receptor antagonistaccording to claim 1, having a structural formula as follows:


4. The cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist according to claim 3, having astructural formula as follows:


5. The cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist according to claim 3, having astructural formula as follows:


6. The cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist according to claim 3, having astructural formula as follows:


7. The cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist according to claim 3, having astructural formula as follows:


8. Applications of the cyclopropyl unsaturated quinoline compound or itspharmaceutically acceptable salts, hydrates, solvates, or prodrugs usedas leukotriene receptor antagonist according to claim 1 in preparing adrug for treating and/or preventing and/or delaying and/or providingadjuvant therapy for asthma and/or allergic rhinitis and asthmasyndromes.
 9. A drug composition, characterized in that comprising thecyclopropyl unsaturated quinoline compound or pharmaceuticallyacceptable salts, hydrates, solvates, or prodrugs used as leukotrienereceptor antagonist according claim
 1. 10. A drug composition accordingto claim 9, further comprising pharmaceutically acceptable excipient(s).